The present invention relates to a functional screen assay for nonsense or frameshift mutations that allows genes of interest to be scanned. More specifically, this invention relates to an assay for the rapid screening for a number of genetic diseases which involves the cloning of segments of a gene in-frame with a sequence coding for a marker and determining the expression level of the marker.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases to provide additional details respecting the practice of the invention, are incorporated by reference. For convenience, the references are referenced numerically in the following text and grouped respectively in the appended bibliography.
The accurate detection of mutation in human genes is important for the clinical diagnosis of inherited diseases and diagnosis of predisposition to neoplasia. Linkage analysis can follow the inheritance of these mutant genes within kindreds with a calculated likelihood for carrier status, but only a direct analysis of the gene will determine the genotype of an at-risk individual in the absence of familial analysis. Therefore, there is a need for rapid and reliable methods for detecting mutations in human genes, especially in human tumor suppressor genes.
One human suppressor gene that represents an excellent candidate for rapid detection of mutations is adenomatous polyposis coli (APC). APC is an inherited syndrome that predispose affected individuals to colon cancer and other types of neoplasia. APC is transmitted in families as an autosomal dominant disease and is caused by mutations in the APC gene. Disruption of the APC gene leads to a highly penetrant, inherited form of colon cancer.
The distribution of germline mutations in APC has been particularly revealing. A number of different research groups have identified disease-causing germline mutations in APC. Results of most of these efforts have been summarized by Burt and Groden (1). All mutations identified to date, with the exception of five missense mutations (one of which does not segregate with the disease), are chain-terminating alterations, such as frameshifts and premature termination codons; most of these mutations are unique. Germline mutations have been detected throughout the coding elements of the gene, with some notable exceptions: no mutations have been detected in exons 1 or 2, nor, as yet, in any of the four recently identified exons upstream of exon 1; nor have any mutations been detected in a large segment of exon 15 from nucleotides 4791 to 7932, which suggests that mutations in these regions might be innocuous or lethal.
Mutational analyses of colorectal adenomas and carcinomas at the APC locus have shown that most, if not all, tumors carry at least one nonsense or frameshift APC mutation. (2-5) Many of the tumors analyzed carry two APC mutations or show a loss of heterozygosity affecting the APC region of chromosome 5. These same mutational analyses of tumors also have suggested that over 70% of APC mutations occur within a 3-kb region of exon 15. The fact that this region is contained within the 6.5-kb contiguous genomic sequence of open reading frame suggests that it may be possible to use genomic DNA as a PCR template in an assay. The majority of these mutations are unique within a given family, with the exception of two deletions that together represent about 10% of known germline mutations. Because the APC coding sequence is greater than 8.5-kb, it is extremely difficult to scan or sequence this gene rapidly.
Conventional methods of mutation screening, such as single strand conformation polymorphism analysis, denaturing-gradient gel electrophoresis, temperature-gradient gel electrophoresis, direct sequencing, or RNase protection assays, are all labor-intensive, gel-based assays that are normally reliable for DNA segments no longer than about 500 nucleotides. Other methods that are not gel-based, such as allele-specific PCR and the oligo-ligation assay, are directed only toward the identification of known mutations and are not applicable for detection of unknown mutations. These conventional methods to detect gene mutations rely either on the preparation of cDNA or on screening of each exon of a gene individually. For genes that have several exons, often spread over large distances in genomic DNA, preparation of cDNA or fragments of each individual exon can be tedious and add precious time to otherwise rapid screening processes.
However, these prior art methods are not suitable for many human disease genes. In APC screening, for example, these methods would be inefficient because none of the mutations identified to date has been found at a frequency of more than one in ten. APC is a large gene, containing over 8.5 kb of known open reading frame sequence and encoding RNAs over 10 kb in size. It is composed of at least 19 exons, some of which are alternatively spliced.
A functional assay for the detection of p53 mutations in Li-Fraumeni patients has been described in Frebourg et al. (6). In this case, however, it is the function of the gene product itself that is assayed and thus not generally applicable. The p53 gene is a human tumor suppressor gene and mutations in this gene are common in human cancer. As described by Harris (7), p53 mutations are different from those in most other tumor suppressors. For example, Harris describes that the tumor suppressor genes defective in retinoblastoma and adenomatous polyposis coli (APC) are commonly inactivated by nonsense mutations that cause truncation or instability of the protein. But Harris describes that in p53 nearly 90 percent of the mutations are missense mutations that change the identity of an amino acid in the protein.
Thus, there is a need for a rapid assay for nonsense and frameshift mutations in the diagnosis of genetic diseases. The assay of this invention allows rapid detection of mutations in genes of individuals and avoids problems with prior screening methods. The present assay is based on the level of expression of a marker, the coding sequence of which has been joined in-frame to the 3' end of an amplified and cloned DNA segment of the gene of interest.